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- EMDB-8417: Time-resolved cryo electron microscopy map of the tRNA-bound 30S ... -

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Basic information

Entry
Database: EMDB / ID: EMD-8417
TitleTime-resolved cryo electron microscopy map of the tRNA-bound 30S subunit
Map datatRNA bound 30S subunit
Sample
  • Complex: tRNA-bound 30S subunit
    • Complex: tRNATransfer RNA
    • Complex: 30S ribosomal subunitProkaryotic small ribosomal subunit
    • Complex: mRNAMessenger RNA
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.0 Å
AuthorsFu Z / Kaledhonkar S / Borg A / Sun M / Chen B / Grassucci RA / Ehrenberg M / Frank J
Funding support United States, Sweden, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM55440 United States
Swedish Research Council2015-04682 Sweden
Howard Hughes Medical Institute (HHMI) United States
the Knut and Alice Wallenberg Foundation, RiboCOREKAW 2009.0251 Sweden
CitationJournal: Structure / Year: 2016
Title: Key Intermediates in Ribosome Recycling Visualized by Time-Resolved Cryoelectron Microscopy.
Authors: Ziao Fu / Sandip Kaledhonkar / Anneli Borg / Ming Sun / Bo Chen / Robert A Grassucci / Måns Ehrenberg / Joachim Frank /
Abstract: Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination ...Upon encountering a stop codon on mRNA, polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P-site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or "ribosome recycling," is promoted by the joint action of ribosome-recycling factor (RRF) and elongation factor G (EF-G) in a guanosine triphosphate (GTP) hydrolysis-dependent manner. Here we used a mixing-spraying-based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.
History
DepositionOct 4, 2016-
Header (metadata) releaseOct 19, 2016-
Map releaseNov 23, 2016-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8417.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationtRNA bound 30S subunit
Voxel sizeX=Y=Z: 1.25 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.021817055 - 0.09109498
Average (Standard dev.)-0.00010295666 (±0.005746497)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 480.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.251.251.25
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z480.000480.000480.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-163-114-126
NX/NY/NZ210124170
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0220.091-0.000

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Supplemental data

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Sample components

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Entire : tRNA-bound 30S subunit

EntireName: tRNA-bound 30S subunit
Components
  • Complex: tRNA-bound 30S subunit
    • Complex: tRNATransfer RNA
    • Complex: 30S ribosomal subunitProkaryotic small ribosomal subunit
    • Complex: mRNAMessenger RNA

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Supramolecule #1: tRNA-bound 30S subunit

SupramoleculeName: tRNA-bound 30S subunit / type: complex / ID: 1 / Parent: 0

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Supramolecule #2: tRNA

SupramoleculeName: tRNA / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: 30S ribosomal subunit

SupramoleculeName: 30S ribosomal subunit / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600

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Supramolecule #4: mRNA

SupramoleculeName: mRNA / type: complex / ID: 4 / Parent: 1
Details: Encodes peptide fMet-Phe-Thr (sequence GGGAAUUCGGGCCCUUGUUAACAAUUAAGGAGGUAUUAAAUGUUUACGUAAUUGCAGAAAAAAAAAAAAAAAAAAAAA)
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.7 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 31000
Specialist opticsSpherical aberration corrector: None / Chromatic aberration corrector: None / Energy filter - Name: None
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-50 / Average exposure time: 0.2 sec. / Average electron dose: 1.01 e/Å2
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND3
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 5241

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